Imaging of anthrax intoxication in mice reveals shared and individual functions of surface receptors CMG-2 and TEM-8 in cellular toxin entry

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8- and CMG-2-dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.

Bacillus anthracis lethal toxin and edema toxin are binary toxins that consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding and cytoplasmic translocation of LF and EF. However, the distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals has not been fully elucidated. Herein, we describe an assay to image anthrax toxin intoxication in animals, and we use it to visualize TEM-8-and CMG-2dependent intoxication in mice. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice expressing a red fluorescent protein in the absence of Cre and a green fluorescent protein in the presence of Cre, intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. Using this assay, we found that: (a) CMG-2 is critical for intoxication in the liver and heart, (b) TEM-8 is required for intoxication in the kidney and spleen, (c) CMG-2 and TEM-8 are redundant for intoxication of some organs, (d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and (e) CMG-2 is the dominant receptor on leukocytes. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for clinical development of reengineered toxin variants for cancer treatment.
Anthrax is contracted through inhalation, ingestion, or cutaneous inoculation of endospores of the Gram-positive bacterium Bacillus anthracis. Spores germinate following their introduction to the body and disseminate to cause a systemic infection, which, if left untreated, is associated with high mortality rates. Upon the death of the host, Bacillus anthracis forms spores that are resistant to chemical insults, heat exposure, and dehydration and remain infectious for long periods (1,2).
The virulence of Bacillus anthracis results from the release of three proteins into the circulation: protective antigen (PA), lethal factor (LF), and edema factor (EF). These three proteins are individually nontoxic, and PA combines with either LF to form anthrax lethal toxin or with EF to form anthrax edema toxin. The systemic administration of anthrax toxin to animals closely mimics experimental infection with Bacillus anthracis, and vaccination against the toxin components is protective, indicating that anthrax is largely a toxin-mediated disease (1,2). Anthrax toxins exert their cytotoxic actions in a threestep activation process that involves: (a) the binding of PA to the surface of target cells, (b) the translocation of LF and EF to the cytoplasmic compartment of the target cells, and (c) the enzymatic action of LF and EF on cytoplasmic substrates (1,2). Anthrax toxin intoxication is initiated by the binding of PA to either of two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8) (3,4). Subsequently, PA is cleaved at the sequence, 164 RKKR 167 , by cell surface-localized furin or furin-like proprotein convertases (5). This endoproteolytic cleavage is absolutely required for toxin activation and triggers all subsequent steps of the intoxication process. The C-terminal 63-kDa fragment of PA (PA63) remains bound to the cell surface after endoproteolytic cleavage and undergoes a conformational change that leads to the formation of a PA63 heptamer or octamer that subsequently binds up to four molecules of LF or EF with high affinity (6,7). The complex of PA63 and LF or EF is then endocytosed, and PA63 undergoes pH-induced conformational changes in the endosomal/lysosomal compartment to form a channel that facilitates the unfolding and translocation of LF and EF to the cytoplasm. EF is an adenylate cyclase proposed to lead to the formation of supraphysiological intracellular levels of cyclic AMP (8). LF is a zinc-dependent metalloproteinase that can cleave and inactivate several mitogen-activated protein kinase kinases, NLR family pyrin domain containing 1 (NLRP1), and regulatory subunits of phosphoinositide-3 kinase (PI3K)-p85α (PIK3R1) and p85β (PIK3R2) (9)(10)(11). Although required for intoxication, the cellular distribution of CMG-2 and TEM-8 and the function of each receptor in the intoxication in specific organs remain to be fully elucidated. Notably, assays for direct visualization of anthrax toxin intoxication in vivo are not available, and the tissue and cellular targets for anthrax toxin during in vivo infection have been inferred only indirectly from analysis of tissues from intoxicated animals or from biochemical and genetic analysis of anthrax toxin targets (12,13).
LF is stable in circulation when administered alone and only becomes cell surface-associated after the binding of PA to CMG-2 or TEM-8 and its subsequent proteolytic cleavage to PA63. It has long been noted that LF residues 1 to 254 suffice to achieve translocation of a variety of "passenger" polypeptides and other molecules into the cytoplasm of the cells in a PA63-dependent manner (14,15). These include other bacterial toxins and bacterial proteins (14,(16)(17)(18)(19)(20), fluorescent proteins (21), viral proteins (22)(23)(24)(25), eukaryotic proteins (26)(27)(28)(29), and radioisotopes (30,31). Thus, the fusion or conjugation of LF to imageable moieties could provide ideal agents for studying the cellular intoxication by anthrax toxin in vivo. A significant caveat to this approach, however, is the low number of LF molecules successfully translocated to the cytoplasm through the PA pore, which makes most imaging modalities poorly suited to study anthrax toxin intoxication in whole-animal systems (19,28). A second challenge to wholeanimal imaging approaches is that most imaging modalities, such as radionuclides, enzymes such as horseradish peroxidase and β-galactosidase, and fluorescent proteins, likely would not discriminate between productive intoxication (i.e., PAdependent entry into the cytoplasm) and nonproductive interactions of the labeled toxin with cells, such as cell surface retention, fluid phase pinocytosis, and endosomal/lysosomal accumulation of intact or partially degraded toxin conjugates.
Spleen extracts from reporter mice carrying a Cre-activated β-galactosidase transgene have been shown to express increased β-galactosidase activity when infected with Salmonella enterica serovar Typhimurium carrying the type III secreted protein, SopE, fused to bacteriophage P1 Cre recombinase (32). Although single-cell resolution was not achieved, presumably due to a low number of cells being infected (32), the study provided evidence that bacterial protein-Cre fusion proteins may display sufficient enzymatic activity in animals to induce LoxP-dependent recombination.
In this study, we used a combined biochemical and genetic approach to image anthrax toxin intoxication in animals. Specifically, we generated a tripartite fusion protein that consists of the N-terminal domain of LF fused to a nuclear localization signal-tagged bacteriophage P1 Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were coadministered to transgenic mice that ubiquitously express a red fluorescent protein (tdTomato) in the absence of Cre activity and a green fluorescent protein (eGFP) in the presence of Cre activity (hereafter mTmG mice), anthrax toxin intoxication could readily be visualized at single-cell resolution by using confocal microscopy of unfixed and unprocessed organs or by flow cytometry. By superimposing individual or combined genetic deficiency of either TEM-8 or CMG-2 in mTmG mice, we were able to directly establish the importance of each receptor in anthrax toxin intoxication in individual tissues.
The assay presented here should be useful for basic and clinical/translational studies of Bacillus anthracis infection, and it may be easily adapted to image intoxication in animals by related bacterial type III toxins. Furthermore, the assay will be valuable for identifying on-and off-targets for reengineered anthrax toxins currently in clinical and preclinical development for the treatment of solid tumors (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48). When used in conjunction with modified PA variants that are activated by specific cell surface proteases, the assay may also be suitable for in vivo imaging of specific cell surface proteolytic activity in a variety of physiological and pathological processes.

Results
Generation of LFn-Cre recombinase fusion proteins capable of PA-dependent cytoplasmic translocation We have previously shown that PA-dependent translocation of a LF-β-lactamase fusion protein can be imaged in cultured cells by using a cell-penetrating β-lactamase quenched fluorescence resonance energy transfer substrate (19). The adaptation of this assay for imaging anthrax toxin intoxication in whole animals, while hypothetically feasible, is prohibited by the high cost of the β-lactamase substrate and, likely, by logistic problems associated with systemic delivery of the substrate to animals. We therefore explored the possibility of combining biochemical and genetic approaches to imaging anthrax toxin intoxication in whole animals. Specifically, we generated a series of proteins that consist of the PA-binding domain of LF (LFn) either fused directly to the bacteriophage P1 Cre recombinase (LFn-Cre) or linked via a nuclear localization signal (LFn-NLS-Cre), a native ubiquitin protein (LFn-native ubiquitin-NLS-Cre), or a modified ubiquitin protein followed by a nuclear localization signal (LFn-modified ubiquitin-NLS-Cre) in which the seven Lys residues of ubiquitin are replaced with Arg. This modified ubiquitin also contains the substitutions V5A and V17A chosen to destabilize the N-terminal region and facilitate translocation through the PA channel (Fig. 1, A and B). The inclusion of ubiquitin as a linker was based on the expectation that ubiquitin fusions of this type, once delivered to the cytosol, would be cleaved by deubiquitinating enzymes, releasing the Cre domain as a smaller entity that may have greater access to the nucleus, the location of its target DNA substrate (49). Furthermore, release from LFn could decrease the probability that ubiquitination of the LFn region would deliver the entire fusion protein to the proteasome for degradation. Similarly, the replacement of the seven Lys residues of ubiquitin with Arg in LFn-modified ubiquitin NLS-Cre could limit ubiquitination and increase stability.
Preliminary testing of the four purified LFn-Cre proteins was done in CV1-5B reporter cells, which contain a Cre-activated β-galactosidase gene, by treating the cells with increasing concentrations of fusion protein in the presence of a fixed concentration of PA ( Fig. 1, C-E). These experiments identified LFn-NLS-Cre as the most effective of the four fusion proteins.
The need for an NLS, the only feature present in LFn-NLS-Cre compared with LFn-Cre (Fig. 1A), suggests that the NLS reported to exist in Cre (50) is not by itself sufficient to maximize nuclear delivery in this context. The alternative explanation that the added NLS in LFn-NLS-Cre provides a target by which cytosolic proteases release the Cre domain cannot be discounted without further study. The total inactivity of LFn-native ubiquitin-NLS-Cre compared with that of LFn-modified ubiquitin-NLS-Cre indicated that native ubiquitin is unable to translocate through the PA pore. The entry of LFn-NLS-Cre into cells was highly PA-dependent, with very few CV1-5B cells displaying β-galactosidase activity in the absence of PA, even at high concentrations of the fusion protein (Fig. 1D).

Imaging anthrax toxin intoxication in mice
The above studies showed that LFn-Cre fusion proteins could translocate to the cytoplasm in a PA-dependent manner that the Cre moiety (alone or as an intact fusion protein) thereafter was imported to the nucleus, and that it retained its recombinase activity after nuclear translocation. This indicated that LFn-Cre fusion proteins, when used in conjunction with appropriate transgenic mouse Cre reporter strains, could be suitable for imaging anthrax toxin intoxication in vivo. The B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J transgenic mouse (hereafter mTmG) is a widely used reporter strain for Cre activity (51). The strain constitutively expresses a floxed plasma membrane-tagged tdTomato (mTomato) fluorescent protein gene under the control of a cytomegalovirus-β-actin enhancerpromoter. This gene is excised by Cre, which simultaneously allows for transcription of a plasma membrane-tagged enhanced green fluorescent protein (eGFP)-expressing gene by placing it proximal to the cytomegalovirus-β-actin enhancerpromoter ( Fig. 2A). Thus, following cytoplasmic translocation of LFn-NLS-Cre through the PA pore and subsequent nuclear Only select molecular weight marker bands (kDa) are indicated due to space limitations. C and D, CV1-5B cells harboring a single copy of lox-stop-lox (Cre-activated) β-galactosidase gene were incubated with varying concentrations of LFn-NLS-Cre and PA as indicated for 40 h. Cells were then stained in situ for β-galactosidase activity using the chromogenic substrate, 5-bromo-3-indolyl β-D-galactopyranoside (Bluo-Gal) as described in Experimental procedures. In panel D, L is LFn-NLS-Cre and P is PA. Numbers are the nanomolar concentration of each component. Images collected with 10× objective. Scale bar is 100 μm. E, CV1-5B cells were plated at low density (left panel) or high density (right panel) and incubated with increasing concentrations of LFn-NLS-Cre, LFn-Cre, LFn-native ubiquitin-NLS-Cre, or LFn-modified ubiquitin-NLS-Cre fusion proteins in the presence of 12 nM PA. Cells were stained for β-galactosidase activity after 40 h using chlorophenol red-β-D-galactopyranoside (CPRG), as described in Experimental procedures. LFn-NLS-Cre, LF fused to a nuclear localization signal-tagged Cre recombinase; PA, protective antigen. Visualizing anthrax toxin intoxication in animals import, intoxicated cells should display green fluorescent plasma membranes, while plasma membranes of nonintoxicated cells should display only red fluorescence.
To test if PA and LFn-NLS-Cre could mediate LoxPdependent recombination in a whole-animal context, we first designed PCR primer sets that would selectively amplify, respectively, the nonrecombined and the recombined mTmG transgene ( Fig. 2A). Interestingly, a PCR product derived from the recombined transgene was readily detected in the heart, lungs, liver, kidney, spleen, lymph nodes, thymus, uterus, esophagus, trachea, tongue, and bone marrow of mTmG +/ 0 mice injected with LFn-NLS-Cre and PA but not in these organs from noninjected mTmG +/0 mice (Fig. 2B). This PCR product was not observed in the intestine, skin, and brain of mice injected with LFn-NLS-Cre and PA (Fig. 2B).
We next determined the ability to detect fluorescence in mTmG +/0 mice by confocal microscopy of unfixed whole organ slices, which could serve as a convenient readout for Cre activity. Wild-type mice were analyzed in parallel as a control for autofluorescence. To obtain semiquantitative estimates of fluorescence intensities of the examined organs, in this and the following experiments, we generated composite images of the entire organ slice from confocal images acquired at low magnification. As expected, red fluorescence of variable intensity was observed in multiple organs of mTmG +/0 mice but not in the corresponding organs from wild-type mice imaged using the identical conditions (Fig. S2). This indicated that the mTmG mouse strain should be suitable for the detection of anthrax toxin intoxication in cells in a variety of organs and tissues by this simple procedure.
Having established that fluorescence in mTmG +/0 mice can readily be detected by confocal microscopy of unfixed whole organ slices, we next intravenously injected mTmG +/0 and wild-type mice with LFn-NLS-Cre alone, PA alone, or with PA in combination with LFn-NLS-Cre (Fig. S3). Mice were killed 24 h later, and slices of excised organs were immediately examined by confocal microscopy. Organs from mTmG +/ 0 mice injected with LFn-NLS-Cre alone displayed only red fluorescence (Fig. S3, A, A', D, D', G, G', J, J', M and M'), importantly showing that the LFn-NLS-Cre protein by itself does not gain access to cells. As expected, organs from mice injected with PA alone also displayed only red fluorescence (Fig. S3, B, B', E, E', H, H', K, K', N and N'). Interestingly, however, when analyzing these organs from mTmG +/0 mice injected with LFn-NLS-Cre in combination with PA, both red ( Fig. S3, C, F, I, L and O) and green fluorescence were readily detected in the heart, kidney, liver, lungs, and spleen (Fig. S3, C', F', I', L' and O'), indicating that PA-dependent translocation of LFn-NLS-Cre to the cytoplasm of cells in these organs takes place and that the subsequent nuclear import of the fusion protein leads to the excision of the floxed mTomato gene. No green fluorescence was observed in the same organs of wildtype mice injected with LFn-NLS-Cre in combination with PA, further demonstrating the origin of the green fluorescent signal from de novo expression of eGFP (Fig. S4, compare A with B, C with D, E with F, G with H, I with J). Compatible with the PCR analysis, green fluorescence was weak or absent in the intestine (Fig. S5, A and B), skin (Fig. S5, C and D), and brain (Fig. S5, E and F) slices, although cells in these organs of mTmG +/0 mice were previously shown to undergo recombination in vivo and express eGFP in the presence of a Creexpressing transgene (51).
Effect of route of administration, dose, and time of analysis on imaging of intoxication Parameters affecting the intoxication process and the optimal use of mTomato/eGFP fluorescence as a readout for anthrax toxin intoxication would be expected to include the mode and dose of toxin delivery, tissue accessibility, receptor expression, efficiency of cytoplasmic translocation and nuclear transport of the LFn-NLS-Cre fusion protein, rate of Cremediated recombination, decay rate of the mTomato mRNA and protein, the rate of transcription, translation and attainment of steady state levels of the eGFP protein, ploidy of the target cells (diploid versus polyploid, carrying one or more than one transgene), and time of imaging after toxin administration. The next set of experiments were aimed at empirically addressing some of these issues by varying the mode and dose of toxin delivery, number of injections, and time from injection to imaging. We first compared low-magnification confocal images of green fluorescence obtained from whole organ slices of the heart, kidney, liver, lungs, and spleen from mTmG +/0 mice injected either intravenously or intraperitoneally with PA in combination with LFn-NLS-Cre (Fig. S6). This analysis showed that toxin administered intraperitoneally intoxicated the organs less efficiently, as evidenced by less green fluorescence, than when administered intravenously (Fig. S6, compare A-C with D, E-G with H, I-K with L, M-O with P, Q-S with T).
We next studied the effect of multiple injections of PA and LFn-NLS-Cre on the intoxication in the five organs analyzed above (Fig. S7). Specifically, we injected mTmG +/0 mice with PA and LFn-NLS-Cre at 0 h, 24 h, 48 h, and 72 h and procured low-magnification red and green confocal fluorescence images of organ slices at 24 h, 48 h, 72 h, and 120 h. For each time point analyzed, a separate control group of mice injected with PA and LFn-NLS-Cre once at 0 h was included to control for Cre-mediated recombination occurring during the assay period and/or the eGFP protein not having attained steadystate levels at 24 h. This control experiment, indeed, showed that the eGFP signal in organs of mice having received a single injection of PA and LFn-NLS-Cre increased qualitatively beyond 24 h, with the possible exception of the liver (Fig. S7, compare A' with B', C', and D', E' with F', G', and H', I' with J', K', and L', M' with N', O', and P', Q' with R', S', and T'). However, for the kidney (Fig. S7, compare F' with F''', G' with G''', H' with H'''), liver (Fig. S7, compare J' with J''', K' with K''', L' with L'''), and spleen (Fig. S7, compare R' with R''', S' with S''', T' with T'''), an increased eGFP signal was evident in mice receiving multiple injections of PA and LFn-NLS-Cre, as compared with single-injected control mice. Interestingly, the mTomato signal in the liver was greatly diminished already at 48 h in mice receiving either single or multiple injections of PA

Visualizing anthrax toxin intoxication in animals
and LFn-NLS-Cre (Fig. S7, compare I with J and J''). This indicates that intoxication in the majority of cells in this organ was achieved.
To determine when an eGFP signal is first detectable after the administration of PA and LFn-NLS-Cre, we injected mTmG +/0 mice with 75 μg of each protein and examined the heart, kidney, liver, lungs, and spleen by confocal microscopy at 6, 8, 10, and 12 h (Fig. S9). Whereas no signal was observed at any of these time points in the heart (Fig. S9, A' (Fig. S9L').
We next surveyed a wide range of organs for anthrax toxin intoxication by intravenously injecting mTmG +/0 mice at 0 h, 24 h, 48 h, and 72 h with either PBS or 25 μg LFn-NLS-Cre and 25 μg PA, followed by analysis of tissues by confocal microscopy at 120 h (Fig. 2C). As found in the prior experiments, intoxication was easily detected in the heart, lung, liver, kidney, and spleen. In addition, intoxication was observed in the esophagus and bone marrow, whereas intoxication of the lymph nodes, thymus, intestine, uterus, trachea, tongue, skin, and brain was not observed.

High-resolution imaging of anthrax toxin intoxication
Using the knowledge gained from the above experiments, we next tested the ability of the assay to image intoxication in individual cells in unprocessed organs. For this purpose, mice received three intravenous injections of 25 μg LFn-NLS-Cre and 25 μg PA at 0 h, 24 h, and 48 h. Seventy-two hours after the first injection, confocal images of blue (nuclei), red (tdTomato), and green (eGFP) fluorescence of slices of the heart, kidney, liver, lungs, and spleen were acquired at high magnification (Fig. 3). This analysis showed that in tissues of these five organs, nonintoxicated and intoxicated individual cells were readily distinguishable by their red and their green membrane-confined fluorescence, respectively.

, D-D''', H-H''' and J-J''').
We next performed a similar comparison of toxin-treated mTmG + and Tem8 −/− ;mTmG + mice analyzed in parallel. This showed that, unlike CMG-2, TEM-8 was not critical for the intoxication of the heart and liver (Fig. 5, B-B''' and F-F'''), revealing CMG-2 as the dominant receptor for the intoxication in these organs. Intoxication in the lungs was not substantially affected by the loss of TEM-8 (Fig. 5, H-H'''), suggesting that intoxication in this organ is mediated by either of the two anthrax toxin receptors. In sharp contrast, the loss of TEM-8 markedly reduced the green fluorescent signal in the kidney (Fig. 5, D-D''') and in trabecular structures of the spleen (Fig. 5, J-J'''), suggesting that TEM-8 plays a key role in the intoxication in these organs. Neither green nor red fluorescence was observable in areas of the spleen harboring leukocytes. However, both nonintoxicated, red fluorescent and intoxicated green fluorescent spleen leukocytes were readily detected by flow cytometry (see below).
The above studies revealed minimal effect of individual deficiencies in either CMG-2 or TEM-8 on the intoxication of lungs. To exclude that this was a consequence of hitherto unrecognized anthrax toxin receptors, we imaged intoxication of Cmg2 −/− ;Tem8 −/− ;mTmG + mice (Fig. 6). This analysis showed that the combined elimination of CMG-2 and TEM-8 completely prevented intoxication of all tissues examined.
To determine the role of TEM-8 and CMG-2 in intoxication of leukocytes, single-cell suspensions of CD45-positive leukocytes from spleens were analyzed by flow cytometry. About half of leukocytes from mTmG + spleens displayed red fluorescence indicating expression of the transgene (Fig. 7A, left  panel). Less than 2% of the leukocytes expressed green fluorescence, showing that intoxication of spleen leukocytes is inefficient (Fig. 7A, right panel). No significant reduction in the percentage of green fluorescent leukocytes was seen after elimination of TEM-8 (Fig. 7A, right panel). However, loss of CMG-2 resulted in a fivefold reduction in the percentage of green fluorescent leukocytes, revealing CMG-2 as the dominant receptor (Fig. 7A, right panel). When analyzing individual leukocyte subsets, this predominance of CMG-2 in intoxication was seen in B cells (Fig. 7B, red bars), T cells (Fig. 7B, yellow bars), neutrophils (Fig. 7B, green bars), and dendritic cells (Fig. 7B, blue bars), whereas monocytic cells were unaffected by single loss of either TEM-8 or CMG-2 (Fig. 7B, purple bars).

Discussion
The ability to directly visualize the intoxication in cells by bacterial exotoxins in whole-animal systems is desirable in the context of enhancing the understanding of the pathogenesis of these toxins and exploring their uses as therapeutics. The development of such imaging assays is complicated by the often low numbers of toxin molecules targeting individual cells, making toxin detection challenging, as well as by the difficulty of providing sufficient spatial resolution to discriminate bona-fide intoxication from nonproductive association of the toxin with the target cell, such as cell surface retention or endosomal/lysosomal sequestration.
Here, we describe a novel assay that provides single-cell resolution imaging of anthrax toxin intoxication in animals. The assay takes advantage of the potential of genetic readout Visualizing anthrax toxin intoxication in animals systems to provide near-unlimited signal amplification, in this case, entailing the activation of a fluorescent reporter gene by an anthrax toxin lethal factor-Cre fusion protein that is translocated to the cytoplasm through the anthrax protective antigen heptamer or octamer pore and subsequently imported to the nucleus. Intoxication in some organs could be detected as early as 12 h after systemic introduction of the toxin, and as expected, the efficiency of intoxication varied with toxin dose and mode of administration. The plasma membrane illumination provided by the membrane-targeted eGFP and tdTomato proteins, when combined with the staining of nuclei obtained by the in vivo Hoechst labeling procedure, provided sufficient cellular and tissue morphology information to allow for the identification of intoxicated cells in unfixed and unprocessed organ slices. It should also be noted that although only excised organs were examined here, the assay should be adaptable for multiphoton intravital imaging of cellular intoxication with essentially no modifications needed.
In a broad survey of organs, intoxication was documented by confocal microscopy in the heart, lung, liver, kidney, spleen, esophagus, and bone marrow, whereas intoxication of the lymph nodes, thymus, intestine, uterus, trachea, tongue, skin, and brain was not observed. The latter tissues may not be exposed to sufficient toxin to allow for cellular entry and/or Cre-mediated recombination and/or may express no or insufficient CMG-2 and TEM-8. Alternatively, the confocal sections analyzed may not have captured the target cell population of a particular organ. In support of the latter, for five of the tissues in which intoxication was not observed by confocal microscopy, intoxication nevertheless could be detected by PCR.
The development of our novel imaging assay afforded the first opportunity to directly visualize CMG-2-and TEM-8- Mice were injected as above and analyzed by flow cytometry after staining with markers for each leukocyte sub-population. Results are shown as mean ± sem. Left panel shows compiled data from all leukocyte subsets. The remaining panels show individual data from each leukocyte subset in toxin-injected wild-type, PBS-injected mTmG mice, toxin-injected mTmG mice, toxin-injected TEM-8-deficient mTmG mice, and toxin-injected CMG-2-deficient mTmG mice. Data are compiled from four separate experiments with 4 to 7 mice per treatment group and genotype. CMG-2, capillary morphogenesis protein-2; LFn-NLS-Cre, LF fused to a nuclear localization signal-tagged Cre recombinase; TEM-8, tumor endothelial marker-8. mediated anthrax toxin intoxication and thereby enhance our understanding of the two receptors in the intoxication process. Intoxication was dependent on expression of either CMG-2 or TEM-8, as mice with combined deficiency in the two anthrax toxin receptors were refractory to intoxication in all organs analyzed. Previous genetic studies in mice have demonstrated that CMG-2 deficiency conferred far greater resistance to anthrax lethal toxin and B. anthracis spore exposure than TEM-8 deficiency and identified the heart and liver as key CMG-2-dependent targets for, respectively, anthrax lethal toxin and anthrax edema toxin (12,52). Assuming that both receptors were close to ubiquitously expressed in tissues, this was tentatively suggested to be a consequence of a more than tenfold lower affinity of PA for TEM-8 than for CMG-2. These findings are compatible with the current imaging study, showing that TEM-8 was essentially unable to support the intoxication in the heart and liver, despite repeated toxin exposure through multiple injections. It should be noted, however, that full intoxication in other organs, including the spleen and kidney, was dependent upon TEM-8. This unequivocally demonstrates that TEM-8 is a functional receptor for anthrax toxin in vivo despite its reported lower affinity for PA and the reported primary importance of CMG-2 in wholeanimal intoxication (12,52).
A curious discrepancy between the aforementioned genetic studies and our current imaging study pertains to the intestine: Genetic studies have definitively established intestinal epithelial cells as direct targets for anthrax edema toxin (12). Nevertheless, we were consistently unable to observe the intoxication in intestinal epithelium. Intestinal epithelial cells of the mTmG +/0 reporter mice have previously been shown to undergo recombination in vivo and express eGFP in response to constitutive or inducible Cre expression, showing that the mTmG transgene is not inherently refractory to Cre-mediated recombination in intestinal epithelium (51). An attractive explanation for this discrepancy pertains to the lack of toxicity of LFn-NLS-Cre/PA as compared with edema toxin. We speculate that edema toxin may initially be unable to access this barrier tissue, but because damage to other visceral organs progresses as a consequence of edema toxin intoxication, endothelial and/or epithelial barrier breakdown may allow entrance to the intestinal epithelial cells.
The imaging assay described here is simple and robust, and importantly, it does not require the handling of toxic proteins. Therefore, it should be amenable for and adaptable to diverse research settings. The assay should be useful for answering a number of basic research questions regarding the pathogenicity of anthrax toxins, as well as assisting clinical/translational efforts aimed at optimizing the treatment of individuals accidentally or deliberately exposed to Bacillus anthracis.
A potential limitation of the novel assay is that the Cre/LoxP system only allows for a binary readout as regards individual cells (nonintoxicated/intoxicated). In a whole-organ context, this should not prevent the generation of semiquantitative or quantitative data, as shown by the relationship between the toxin dose administered and the fluorescence intensity observed in individual organs. For such quantitative studies, the fraction of intoxicated cells in a given tissue could be enumerated by confocal image analysis. Alternatively, the ratio of green to red fluorescence could be determined in wholeorgan extracts.
Considerable effort is currently being expended on the development of modified anthrax toxins as novel agents for the treatment of human malignancies. Strategies employed include the reengineering of PA to bind tumor cell surface-enriched proteins (33,34), and the reengineering of PA to be proteolytically activated by proteases enriched in the tumor microenvironment, including matrix metalloproteinases (35)(36)(37)(38)(39)(40)(41)(42), urokinase plasminogen activator (37,39,41,44,47,53), and testisin (48). The assay described here is eminently suited for assessing the efficiency of LF delivery to tumor-relevant cell populations by these modified PAs, as well as to systematically delineate off-targets, which may be invaluable for dose and route of delivery optimization. Last, but not least, by using PA variants selectively cleaved by specific cell surface proteases (42,45,48), the assay may be used for in vivo imaging of specific cell surface proteolytic activity in diverse physiological and pathological settings.

Recombinant proteins
Plasmids for expressing proteins having LFn (LF aa 1-254) fused to Cre recombinase were constructed using the Champion pET SUMO vector (Invitrogen), which expresses proteins fused at the C-terminus of a His6-SUMO tag. DNA-encoding residues 1 to 254 of anthrax lethal factor originated from Bacillus anthracis, and those of Cre recombinase from bacteriophage P1. The sequences of the four LFn-Cre proteins are shown in Fig. S1. They differ only in the linker sequences. Two of the constructs contain ubiquitin between LFn and Cre, and three contain a nuclear localization signal (NLS) preceding Cre. The proteins were expressed as per the Champion pET SUMO manufacturer's instructions with minor modifications. Cell lysates were adsorbed to Ni-NTA resins, and the Histagged proteins were eluted with imidazole solutions. The His6-SUMO-LFn-Cre proteins were cleaved with SUMO protease, which was made inhouse using Addgene plasmid #64697, deposited to Addgene by Dr Hideo Iwai (54). The His6-SUMO polypeptide and the His6-tagged SUMO protease were subtractively removed by passage through Ni-NTA resin. The LFn-Cre proteins were further purified by chromatography on hydroxyapatite to achieve purities of >95%. The LFn-NLS-Cre protein selected for the animal imaging studies was obtained in yields of >20 mg/l of culture.

Cell culture assays
Efficacy of LFn-Cre protein translocation into cells was tested in CV1-5B cells (55,56), which contain a single copy lox-stop-lox-β-galactosidase transgene. Cells were plated in 12-and 96-well plates in DMEM high-glucose medium with 10% fetal bovine serum, cultured at 37 C at 10% CO 2 , and used when they were at low and high confluency. PA and

Imaging anthrax toxin intoxication in mice
LFn-NLS-Cre and PA proteins alone or in combination in PBS were delivered intraperitoneally or via tail vein injection. The mice were tail vein-injected with 100 μl of 6 mg/ml Hoechst 33342 dye (Thermo Fisher Scientific) 4 to 6 h prior to termination of an experiment to visualize nuclei (58). Mice were euthanized by CO 2 inhalation and perfused with ice-cold PBS using cardiac puncture. Organs were immediately removed and cut into 1 to 2 mm thick slices using a scalpel. The organ slices were placed on a MatTek glass bottom microwell dish (MatTek Corporation) and imaged using a 20× 0.75 NA Air or 60× 1.27 NA Water objective (Nikon) on an A1R + MP confocal microscope system (Nikon). Large images were composed of stitched images with a 10% overlap using NIS-Elements software (Nikon).

Data availability
All data presented and discussed in the article are contained within the body of article and supporting figures.
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